tRF-1:30-Gly-CCC-3 inhibits thyroid cancer via binding to PC and modulating metabolic reprogramming

This study reports a mechanism of tRNA fragments in PTC whereby tRF-30 controls PC abundance and subsequent TCA cycle anaplerosis to affect metabolic reprogramming and cancer progression.


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Full guidelines are available on our Instructions for Authors page, https://www.life-science-alliance.org/authorsWe encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript.If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information.These files will be linked online as supplementary "Source Data" files.***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available.Failure to provide original images upon request will result in unavoidable delays in publication.Please ensure that you have access to all original microscopy and blot data images before submitting your revision.*** This article is an interesting discovery.Through high-throughput sequencing, the author selected tRF-1: 30-Gly-CCC-3 with low expression in thyroid cancer for research and verified that this tRF can inhibit the malignant progression of thyroid cancer through experiments such as cck8 and transwell.The tRF-binding protein PC was found by Pull down assay, and it was found that the tRF could down-regulate the expression of the protein.Finally, the authors found that the tRF can regulate the metabolic reprogramming of PTC by interfering with the TCA cycle.In summary, the author confirmed the role of tRF in PTC and found novel regulatory mechanisms.I recommend accepting the publication of this article, but there are still some small problems that need to be improved before the final publication.1.The authors found that tRF-5c was the main down-regulated type, but did not clearly explain how to directly select tRF-1: 30-Gly-CCC-3 for subsequent verification based on factors such as P-value, FC-value, and length.2. The author selected the two cells with the lowest expression for subsequent experiments, but generally, we will choose the cells with relatively high expression for a knockdown, please explain the reason why TPC1 or NPA87 was not selected for a knockdown.3. The too-long naming of the tRF in Figure affects the cleanliness and beauty, so it is recommended to simplify its naming.4. In Figure 2I, whether the S and N groups are marked reversely.5. Please check the format of the symbols in the text.6.How does this tRF specifically regulate its stability by binding to PC protein? 7. The Warburg effect is an important metabolic feature of tumor cells.The author found that the tRF can reduce the level of several important intermediates in the TCA cycle, and whether the tRF will have an effect on the Warburg effect.
1st Authors' Response to Reviewers November 4, 2023 1 Dear Editor, Thank you very much for providing us with the comments on our manuscript entitled "tRF-1:30-Gly-CCC-3 inhibits thyroid cancer via binding to PC and modulating metabolic reprogramming".We thank the reviewer for the time spent reviewing our manuscript and for the highly constructive and helpful comments that enabled us to further improve the quality of our manuscript.We did our utmost to address all issues that were raised, as listed point-by-point below.

Reviewer #1 (Comments to the Authors (Required)):
This article is an interesting discovery.Through high-throughput sequencing, the author selected tRF-30 with low expression in thyroid cancer for research and verified that this tRF can inhibit the malignant progression of thyroid cancer through experiments such as cck8 and transwell.The tRF-binding protein PC was found by Pull down assay, and it was found that the tRF could down-regulate the expression of the protein.Finally, the authors found that the tRF can regulate the metabolic reprogramming of PTC by interfering with the TCA cycle.In summary, the author confirmed the role of tRF in PTC and found novel regulatory mechanisms.I recommend accepting the publication of this article, but there are still some small problems that need to be improved before the final publication.
1.The authors found that tRF-5c was the main down-regulated type but did not clearly explain how to directly select tRF-1:30-Gly-CCC-3 for subsequent verification based on factors such as P-value, FC-value, and length.
Response: Dear reviewer, thanks for this precious suggestion.As suggested, we have provided raw data on significantly dysregulated tRNA fragments in the Supplementary Materials section in RED as follows: A form of tRF-5c known as tRF-1:30-Gly-CCC-3 has a length longer than 18 bp, a high FC-value of 0.202, and a low p-value of 0.005.Additionally, this tRF has now been included in the Mintbase.After a comprehensive analysis of the aforementioned elements, tRF-1:30-Gly-CCC-3 was selected for subsequent verification.
Table S3: Data of significantly dysregulated tRNA fragments in PTC 2. The author selected the two cells with the lowest expression for subsequent experiments, but generally, we will choose the cells with relatively high expression for a knockdown, please explain the reason why TPC1 or NPA87 was not selected for a knockdown.
Response: Dear reviewer, thank you for the precious suggestion.The expression of tRF-30 was significantly lower in human PTC tissues than in normal tissues, so we selected the two PTC cell lines with the lowest expression of tRF-30, which was similar to the expression pattern of tRF-30 in PTC tissues.Moreover, the knockdown efficiencies of tRF-30 in TPC1 or NPA87 cells were far from satisfactory.The results have been added to the Results section in RED as follows.
We detected the knockdown models of tRF-30 in TPC1 and NPA87 cells by qRT-PCR assay, and the results showed that the knockdown efficiencies of the cells  Response: Dear reviewer, thank you for the precious suggestion.As suggested, we have simplified the long naming of the tRF-1:30-Gly-CCC-3 as tRF-30 in the revised manuscript.
4. In Figure 2I, whether the S and N groups are marked reversely.
Response: Dear reviewer, thank you for the precious suggestion, and we apologize for our oversight.As suggested, we have rewritten the group names in this revised  7. The Warburg effect is an important metabolic feature of tumor cells.The author found that the tRF can reduce the level of several important intermediates in the TCA cycle, and whether the tRF will have an effect on the Warburg effect.
Response: Dear reviewer, thank you for the precious suggestion.As suggested, we further explored whether tRF regulated the Warburg effect.An increase in lactate production and glucose uptake are the two common indexes reflecting the Warburg effect.Therefore, we conducted glucose uptake and lactate production assays, and the results have been added in the Results section in RED as follows: Carcinoma cells show preferential use of lactate-generating glycolysis over the common route of oxidative phosphorylation (OXPHOS).This altered metabolism, named the "Warburg effect", has been considered for a long time to be the major metabolic reprogramming in cancer.The changes in glycolytic pathways have been associated directly or indirectly with the downstream targets of many different ncRNAs (Mirzaei H & Hamblin MR, 2020).Therefore, we further explored whether tRF regulated the Warburg effect.The glucose uptake and lactate production assay results showed that tRF-30 overexpression and knockdown had no significant effect on the glycolysis in BCPAP and KTC1 cells (Fig S3, n.s.P > 0.05).Therefore, we speculated that tRF-30 had no significant effect on the Warburg effect in PTC cells.Thank you for submitting your revised manuscript entitled "tRF-1:30-Gly-CCC-3 inhibits thyroid cancer via binding to PC and modulating metabolic reprogramming".We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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You can contact the journal office with any questions, contact@life-science-alliance.orgAgain, congratulations on a very nice paper.I hope you found the review process to be constructive and are pleased with how the manuscript was handled editorially.We look forward to future exciting submissions from your lab.Sincerely, Eric Sawey, PhD Executive Editor Life Science Alliance http://www.lsajournal.org were unacceptably low (FigS1).Therefore, BCPAP and KTC1 cell lines were chosen for further biological function research and downstream mechanism studies.

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Zhejiang University School of Medicine, Jinhua Department of Breast and Thyroid Surgery, Affiliated Jinhua Hospital, Zhejiang University School of Medicine, Jinhua 321000, Thank you for submitting your Research Article entitled "tRF-1:30-Gly-CCC-3 inhibits thyroid cancer via binding to PC and modulating metabolic reprogramming".It is a pleasure to let you know that your manuscript is now accepted for publication in Life Science Alliance.Congratulations on this interesting work.